Bioartificial extracellular matrix containing hydrogel matrix derivatized with cell adhesive peptide fragment

ABSTRACT

A bioartificial extracellular matrix for use in tissue regeneration or replacement is provided by derivatizing a three-dimensional hydrogel matrix with a cell adhesive extracellular matrix protein or cell adhesive peptide fragment of the protein. Preferably, derivatizing is by covalent immobilization of a cell adhesive peptide fragment having the amino acid sequence, ArgGlyAsp, TyrIleGlySerArg or IleLysValAlaVal. Cartilage or tendon can be regenerated by implanting a matrix containing an adhesive peptide fragment that favors chondrocyte invasion. The matrix can be pre-seeded with cells, and tissue can be reconstituted in vitro and then implanted. A cell-seeded matrix can be encapsulated in a semi-permeable membrane to form a bioartificial organ. An agarose hydrogel matrix having an agarose concentration of 0.5-1.25% (w/v) and an average pore radius between 120 nm and 290 nm is preferred. A nerve guidance channel for use in regenerating severed nerve is prepared containing a tubular semi-permeable membrane having openings adapted to receive ends of a severed nerve, and an inner lumen containing the hydrogel matrix having a bound cell adhesive peptide fragment through which the nerve can regenerate.

This application is a divisional application under 37 CFR §1.53(b) ofU.S. Ser. No. 08/280,646 filed on Jul. 20, 1994, now U.S. Pat. No.5,834,029, incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compositions and methods for a bioartificialextracellular matrix.

BACKGROUND OF THE INVENTION

Tissue engineering in the nervous system deals with the functionalreplacement of damaged tissues and nervous system regeneration.

The ability to organize cells in three dimensions (3-D) is an importantcomponent of tissue engineering. The behavior of cells is influencedboth by their intrinsic genetic programs and their extracellularenvironment. The extracellular environment includes `passive` structuralcomponents and biologically `active` components.

Most cells in multicellular organisms are in contact with an intricatemeshwork of interacting, extracellular macromolecules that constitutethe extracellular matrix (ECM). These macromolecules, mainly proteinsand polysaccharides, are secreted locally and assemble into an organized3-D meshwork in the extracellular spaces of most tissues. ECM moleculesinclude glycosaminoglycans, and proteoglycans such as chrondroitinsulfate, fibronectin, heparin sulfate, hyaluron dermatan sulfate,keratin sulfate, laminin, collagen, heparan sulfate proteoglycan, andelastin. In addition to serving as a universal biological glue, ECMmolecules also form highly specialized structures such as cartilage,tendons, basal laminae, and (in conjunction with secondary deposition ofcalcium phosphate) bone and teeth. Alberts et al., Molecular Biology ofthe Cell, Garland, N.Y., pp. 802-24 (1989).

Extracellular matrices modulate the organization of the intracellularcytoskeleton, cell differentiation and the spatial architecture of cellsand tissues. In act, the ECM plays a critical role in regulating thebehaviour of cells that contact it by influencing cellular development,migration, proliferation, differentiation, shape, polarity and metabolicfunction.

Several peptide active sites responsible for cell attachment have beenidentified in various ECM molecules.

In vivo laminin (LN) immunoreactivity has been detected in severalregions of the embryo including muscles (Chui and Sanes, Dev. Biol.,103, pp. 456-67 (1984)), spinal cord (Azzi et al., Matrix, 9, pp. 479-85(1989), spinal roots (Rogers et al., Dev. Biol., 113, pp. 429-35(1986)), optic nerve (McLoon et al., J. Neurosci., 8, pp. 1981-90(1988)), cerebral cortex (Liesi, EMBO, 4, pp. 1163-70 (1985); Zhou, Dev.Brain Res., 55, pp. 191-201 (1990)), hippocampus (Gordon-Weeks et al.,J. Neurocytol., 18, pp. 451-63 (1989)) and the medial longitudinalfasciculus of the midbrain (Letourneau et al., Development, 105, pp.505-19 (1989)).

The tripeptidic sequence RGD (ArgGlyAsp; AA₂ -AA₄ of SEQ ID NO:2) hasbeen identified to be responsible for some of the cell adhesionproperties of fibronectin (Pierschbacher and Ruoslahti, Science, 309,pp. 30-33 (1984)), laminin (Grant et al., Cell, 58, pp. 933-43 (1989)),entactin (Durkin et al., J. Cell. Biol., 107, pp. 2329-40 (1988)),vitronectin (Suzuki et al., EMBO, 4, pp. 2519-24 (1985)), collagen I(Dedhar et al., J. Cell. Biol., 107, pp. 2749-56 (1987)), collagen IV(Aumailley et al., Exp. Cell Res., 187, pp. 463-74 (1989)),thrombospondin (Lawler et al., J. Cell. Biol., 107, pp. 2351-61 (1988))and tenascin (Friedlander et al., J. Cell. Biol., 107, pp. 2329-40(1988)).

The sequence YIGSR (TyrIleGlySerArg; AA₅ -AA₉ of SEQ ID NO:1), found onthe B1 chain of laminin, promotes epithelial cell attachment (Graf etal., Biochemistry, 26, pp. 6896-900 (1987)) and inhibits tumormetastasis (Iwamoto et al., Science, 238, pp. 1132-34 (1987)).

The IKVAV sequence found on the A chain of laminin, has been reported topromote neurite outgrowth (Tashiro et al., J. Biol. Chem., 264, pp.16174-182 (1989); Jucker et al., J. Neurosci. Res. 28, pp. 507-17(1991)).

All of the studies using these preptidic sequences of cell attachmentand neurite promotion were conducted on flat two-dimension substrates(Smallheiser et al., Dev. Brain Res., 12, pp. 136-40 (1984); Graf etal., Biochemistry, 26, pp. 6896-900 (1987); Sephel et al., Biochem.Biophys. Res. Comm., 2, pp. 821-29 (1989); Jucker et al., J. Neurosci.Res., 28, pp. 507-17 (1991)). The physical and chemical nature of theculture substrate influences cell attachment and neurite extension. Thephysical microstructure of a 2-D culture substrate can influence cellbehavior. The use of permissive and on-permissive culture surfacechemistries facilitates nerve guidance in 2-D. The cell attachmentregulating function of various serum proteins like albumin andfibronectin is dependent on the chemistries of the culture substratesthat they are adsorbed onto.

Gene expression is reported to be regulated differently by a flat 2-Dsubstrate as opposed to a hydrated 3-D substrate. For example, monolayerculture of primary rabbit articular chondrocyte and human epiphysealchondrocyte on 2-D tissue culture substrates causes primary chondrocyteto lose their differentiated phenotype. The differentiated chondrocytephenotype is re-expressed when they are cultured in 3-D agarose gels(Benya and Shaffer, Cell, 30, pp. 215-24 (1982); Aulthouse, et al., InVitro Cell Dev. Bio., 25, pp. 659-68 (1989)).

Similarly, alkaline phosphatase gene expression in primary bile ductularepithelial cells is differentially regulated when they are cultured in3-D Matrigel®, collagen or agarose gels are opposed to 2-D cultures(Mathis et al., Cancer Res., 48, pp. 6145-53 (1988)).

Therefore, 3-D presentation of ECM components may better mimic the invivo environment in influencing cell or tissue response. In particular,in vivo use of ECM biomolecules may require such a 3-D system foroptimal efficacy. The development of a defined, bioartificial 3-D matrixthat presents ECM molecules, or active portions thereof, wouldfacilitate tissue engineering in the nervous system by allowing in vitroand in vivo cell manipulation and cell culture in 3-D. See, e.g., Koebeet al., Cryobiology, 27, pp. 576-84 (1990).

In addition, there is a need to develop a defined, biosynthetic matrix,because the tumorogenic origins of some commercially available ECM,e.g., Matrigel® mouse sarcoma derived ECM, render it unattractive forsome in vitro and in vivo applications. Further, naturally occuring ECMcomponents such as collagen may be enzymatically degraded in the bodywhile a synthetic ECM is less likely to be degraded.

SUMMARY OF THE INVENTION

This invention provides a three-dimensional hydrogel based,biosynthetic, extracellular matrix (ECM) equivalent, and method ofmaking same. Agarose matrices having a chemistry amenable toderivatization with various ECM adhesive peptides and proteins, arepreferred in forming the 3-D hydrogel substrates of this invention.These biologically active 3-D templates may be useful in facilitatingtissue regeneration or replacement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A double Y-axis plot depicting the influence of agarose gelconcentration on average pore radius (Y1) and percent striatal cellsextending neurites (Y2) after 72 hours in culture. Pore radius wascalculated by hydraulic permeability measurements of the different gelconcentrations. Solid line through pore radii data points is anexponential fit with r² =0.985.

FIG. 2. Schematic of an agarobiose unit and the carbonyldiimidazolecoupling chemistry for the immobilization of CDPGYIGSR(CysAspProGlyTyrIleGly SerArg; SEQ ID NO:1) oligopeptide to agarosegels.

FIG. 3. Histogram depicting E9 chick DRG neuritic spread as the ratio oftotal ganglionic spread area/ganglionic cell body area (TGSA/GCBA).Neuritic spread in various gels is plotted as a percentage of neuriticspread in Ag-Plain gels at 2 days (n=6). Error bars represent standarddeviation. `*` depicts a statistically significant higher neuriticspread (p<0.05) relative to Ag-Plain; `#` depicts a statisticallysignificant lower neuritic spread (p<0.05) relative to Ag-Plain.

FIG. 4. Histogram of comparative E9 Chick DRG neurite extension inunderivatized and derivatized agarose gels (n=6). Error bars representstandard deviation. `*` denotes a statistically significant, higherneurite length (p<0.05) compared to Ag-Plain; `#` denotes astatistically significant lower neurite length (p<0.05) compared toAg-Plain.

FIG. 5. Histogram depicting comparative PC12 neuritic extension inunderivatized and derivatized agarose gels (n=6). Error bars representstandard deviation. `*` depicts a significant difference with respect toAg-Plain (p<0.05); `#` depicts a significant difference with respect toall other LN oligopeptide derivatized agarose gels (p<0.05).

FIG. 6. Schematic illustration of dorsal root regeneration across anerve gap of 4.0 mm with a nerve guidance channel whose lumen is loadedwith agarose gel.

FIG. 7. Graph showing the number of myelinated axons regenerated at 4weeks along polymer guidance channels filled with AgPlain andAg-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) gels. "*"depicts a statistically significant difference with p<0.05 using theStudent t test.

FIG. 8. Graph showing the density of myelinated axons regenerated at 4weeks along polymer guidance channels filled with AgPlain andAg-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) gels. "*"depicts a statistically significant difference with p<0.05 using theStudent t test.

FIG. 9. Histogram depicting the number of myelinated axons inregenerating sural nerves at 2.0 mm distance from the proximal nervestump in polymer guidance channels filled with A) saline; B) AgPlain andC) Ag-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1). "*" depictsa statistical difference of p<0.05 when compared to saline or AgPlain.Student t test was used to evaluate statistical significance.

FIG. 10. Histogram depicting the density of myelinated axons inregenerating sural nerves at 2.0 mm distance from the proximal nervestump in polymer guidance channels filled with A) saline; B) AgPlain andC) Ag-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1). "*" depictsa statistical difference of p<0.05 when compared to saline or AgPlain.Student t test was used to evaluate statistical significance.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a biosynthetic, hydrogel-based,three-dimensional bioartificial ECM. The bioartificial extracellularmatrices of this invention offer the possibility of manipulating cellsin 3-D, and may be used as three dimensional templates for tissueengineering efforts in vitro and in vivo.

The term "nerve" means both nonfascicular and polyfascicular nerves.

The term "active factor" or "growth factor" includes any active analogs,active fragments, or active derivatives thereof.

Any suitable hydrogel may be used as the substrate for the bioartificialextracellular matrices of this invention. Compositions that formhydrogels fall into three classes. The first class carries a netnegative charge (e.g., alginate). The second class carries a netpositive charge (e.g., collagen and laminin). Examples of commerciallyavailable extracellular matrix component hydrogels include Matrigel™ andVitrogen™. The third class is net neutral in charge (e.g., highlycrosslinked polyethylene oxide, or polyvinylalcohol).

A hydrogel suitable for use in this invention is preferably a definedpolymer, most preferably a polymer that is synthetic or can be preparedfrom a naturally occurring, non-tumorigenic source, free of undesiredbiological (e.g., bacterial or viral), chemical or other contaminants.Most preferred as the matrix substrate are well characterized hydrogelsthat permit presentation of only the desired ECM adhesion molecule oradhesive peptide fragement in 3-D, substantially free of undesiredadhesion motifs.

Matrigel™ is not a defined substrate and also less desirable since it isderived from a murine sarcoma line. In addition, not all syntheticpolymer hydrogels are suitable. For example, the use of acrylic basedhydrogels by Woerly et al., Cell Transplantation, 2, pp. 229--39 (1993)presents the possibility of cytotoxicity because entrapment of neuronalcells is done concomitantly with the cross-linking reaction in thepresence of free radical initiators.

Polymers that may be useful hydrogel matrix substrate materials includehigh molecular weight polyethylene oxide (PEO) and hyaluronate.Stabilized hyaluronate is commercially available (Fidia AdvancedBiopolymers). Various PEO polymers are also commercially available.

Polysaccharides are a class of macromolecules of the general formula(CH₂ O)_(n) which are useful as the hydrogel substrate in the presentinvention. Polysaccharides include several naturally occuring compounds,e.g., agarose, alginate and chitosan. We prefer agarose.

Agarose is a clear, thermoreversible hydrogel made of polysaccharides,mainly the alternating copolymers of 1,4 linked and3,6-anhydro-α-L-galactose and 1,3 linked β-D-galactose. Two commerciallyavailable agaroses are SeaPrep® and SeaPlaque® agarose (FMC Corp.Rockland, Me.). SeaPrep® is a hydroxyethylated agarose that gels at 17°C. The particular suitability of a hydrogel as a biomaterial stems fromthe similarity of its physical properties to those of living tissues.This resemblance is based on its high water content, soft rubberyconsistency and low interfacial tension. The thermoreversible propertiesof agarose gels make it possible for agarose to be a liquid at roomtemperature allowing for easy mixing of cell-gel solution and thencooling to 4° C. causes gelation and entrapment of cells. This is acomparatively benign process, free of chemicals toxic to the cells.

We prefer an agarose concentration of 0.50 to 1.25% (w/v), mostpreferably 1.0%, for the permissive layers of the hydrogel matrix.

Several physical properties of the hydrogel matrices of this inventionare dependent upon gel concentration. Increase in gel concentration maychange the gel pore radius, morphology, or its permeability to differentmolecular weight proteins.

Gel pore radius determination can be determined by any suitable method,including hydraulic permeability determination using a graduated watercolumn, transmission electron microscopy and sieving spheres of knownradius through different agar gel concentrations. See, e.g., Griess etal., Biophysical J., 65, pp. 138-48 (1993). We prefer hydraulicpermeability-based pore radius determination, as the method mostsensitive to changes in gel concentration.

Measurement of gel hydraulic permeability using a graduated water columnenabled the calculation of average pore radius for each of the gelconcentrations studied. The average gel pore radius fall exponentiallyas the gel concentration increased. The slope of the curve indicated thesensitivity of pore radius to gel concentration. The average gel poreradius preferably varies between 120-290 nm, and is most preferablyapproximately 150 nm. The pore radius of the 1.25% threshold agarose gelconcentration was 150 nm.

The agarose hydrogels of this invention may be used as a carrier topresent various ECM proteins or peptides, e.g., laminin fibronectin,and/or their peptidic analogs in 3-D. The chemistry of agarose permitseasy modification with such ECM adhesive proteins and/or peptides. Weprefer covalent immobilization of ECM proteins to the hydrogel backbone.Such immobilization is important because the physical blending of lowmolecular weight oligopeptides with hydrogels will not retain thepeptides in the gel. Further, covalent immobilization prevents thepossible saturation of cell surface receptors by `free-floating` ECMmolecules in hydrogel-ECM molecule blends.

Any suitable coupling system may be used for derivatization. Mostpreferably, covalent coupling using a bi-functional imidazole couplingagent, e.g., 1'1 carbonyldiimidazole, is used. This coupling chemistrydoes not alter the physical structure of the gel significantly.

The bioartificial hydrogel extracellular matrices of this invention areuseful for presenting in 3-D full length extracellular matrix proteinsinvolved in cell adhesion. In addition, peptide fragments of suchadhesion molecules that contain cell binding sequences may also be used(i.e., adhesive peptide fragement). Several such adhesive peptidefragments are known in the art. A particular peptide fragment can betested for its binding ability or adhesive capacity according tostandard techniques.

The bioartificial hydrogel matrices of this invention can be used topresent ECM adhesion molecules, or adhesive peptide fragments thereof,in 3-D to a variety of cell types. These cell types include any cellthat is normally in contact with the ECM in vivo, or any cell bearing acell surface receptor capable of binding to an ECM adhesion molecule oradhesive peptide fragment thereof.

Useful cells include epithelial cells, endothelial cells, fibroblasts,myoblasts, chondroblasts, osteoblasts, and neural stem cells (Richardset al., PNAS 89, pp. 8591-95 (1992); Ray et al., PNAS 90, pp. 3602-06(1993)). Other cells that may be useful in the methods and compositionsof this invention include, Schwann cells (WO 92/03536), astrocytes,oligodendrocytes and their precursors, adrenal chromaffin cells, and thelike.

Stem cells represent a class of cells which may readily be expanded inculture, and whose progeny may be terminally differentiated by theadministration of a specific growth factor. See, e.g., Weiss et al.(PCT/CA 92/00283).

Myoblasts are muscle precursor cells originally derived from mesodermalstem cell populations, e.g., L-6 and β-CH3 cells. Primary myoblasts canbe readily isolated from tissue taken from an autopsy or a biopsy, andcan be purified and expanded. Myoblasts proliferate and fuse together toform differentiated, multi-nucleated myotubes. Myotubes no longerdivide, but continue to produce muscle proteins. While proliferating,myoblasts may readily be genetically engineered to produce therapeuticmolecules. Myoblasts are capable of migrating and fusing intopre-existing fibers.

It will be appreciated that the choice of ECM adhesion molecule oradhesive peptide fragment for use in the bioartificial ECM matrix willdepend upon the desired target cell type. See, e.g., Kleinman, U.S. Pat.No. 4,829,000; End and Engel, "Multidomain Proteins Of The ExtracellularMatrix And Cellular Growth", in McDonald and Mecham Biology ofExtracellular Matrix Series, Academic Press, NY, pp. 79-129 (1991). Oneof skill in the art can routinely assay and particular ECM molecule oradhesive peptide fragment motif for its adhesive capacity for a chosencell type.

Thus, according to the compositions and methods of this invention, itmay be possible to influence the behavior (i.e., development, migration,proliferation, differentiation, shape, polarity, and/or metabolicfunction) of any ECM-responsive cell type, by providing the appropriateECM-mediated molecular cues.

In some embodiments, the hydrogel ECM matrix can be derivatized with theappropriate ECM adhesion molecules or adhesive peptide fragments andimplanted into a desired location in a host, e.g., a mammal, preferablya human. In these embodiments, the matrix acts as a support for tissueregeneration, whereby the host cells infiltrate the matrix. In thepresence of the appropriate 3-D molecular cues in the matrix host tissueregeneration is facilitated.

Such embodiments have use, for example, in cartilage or tendonregeneration by derivatizing the matrix with ECM adhesion molecules oradhesive peptide fragments that favor chondrocyte invasion. Similarly,the matrices of this invention may be useful in promoting muscle, boneor skin regenerated by presenting the appropriate molecular cues toinfluence myoblast, osteoblast or epithelial cell behavior. In apreferred embodiment, the bioartificial matrices of this invention areused to promote nerve regeneration, in nerve guidance channels.

In other embodiments, the bioartificial matrices of this invention canbe pre-seeded with cells, whereby the cells are suspended in the matrixand exposed to the appropriate molecular cues in 3-D. These cell-seededmatrices are useful in tissue replacement protocols. According to theseembodiments, tissue can be reconstituted in vitro and them implantedinto a host in need thereof. For example, cardiac myoblasts may besuspended in the derivatized hydrogel matrices of this invention tocreate a tissue patch of a thickness corresponding to the cardiac wall.The reconstituted cardiac patch could then be implanted, as part of atissue replacement therapy.

Similar protocols for cartilage, tendon, bone, skin, nerve, bloodvessels and other tissues are contemplated. The ability to casthydrogels, e.g., agarose, into a variety of shapes, as well the abilityto fabricate "permissive" gel concentrations enables the production ofbioartificial matrices that can influence cell behavior in definedplanes or through defined "tracts".

It will be appreciated that according to the foregoing embodiments, thecells may be xenografts, allografts or autografts, preferablyallografts, most preferably, autografts. Surgical procedures forimplanting such cells are known. See, e.g., Gage, U.S. Pat. No.5,082,670.

In other embodiments it may be desirable to encapsulate the cell-seededmatrix in a semi-permeable membrane to form a bioartificial organ. Suchbioartificial organs are well known in the art. See, e.g., WO 92/019195,incorporated herein by reference. In these embodiments the metabolicfunction of the encapsulated cells may be controlled by the ECM adhesionmolecule or adhesive peptide fragment presented in 3-D. The encapsulatedcells can be influenced to produce a biologically active molecule thatmay function within the encapsulated cells, or be released or secretedfrom the encapsulated cells, to exert a therapeutic effect on the host.This allows precise control over cell behavior at a fixed location inthe host body.

In a preferred embodiment, laminin-derived oligopeptidic fragments, anRGD-containing sequence (ArgGlyAsp; AA₂ -AA₄ of SEQ ID NO:2), aYIGSR-containing sequence (TyrIleGlySerArg; AA₅ -AA₉ of SEQ ID NO:1)and/or an IKVAV-containing sequence (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQID NO:3), are coupled to the hydroxyl backbone of agarose, using anysuitable method. Most preferably, the oligopeptidic fragments GRGDSP(GlyArgGlyAspSerPro; SEQ ID NO:2), CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1), or the 19-mer water-solubleamino acid sequence CSRARKQAASIKVAVSADR (CysSerArgAlaArgLysGlnAlaAlaSerIleLysValAlaValSerAlaAspArg; SEQ ID NO:3) are used.

CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) has been shown toevoke only 30% of the maximal response obtained by laminin inchemotactic functions with melanoma cells (Graf. et al., Biochemistry,26, pp. 6896-900 (1987)). Thus, the use of full length ECM molecules mayelicit more significant cellular effects. However, the use of minimaloligopeptides creates a more stringent substrate condition andfacilitates the testing of the gel system without the potent biologicaleffects of full length proteins eclipsing the gel's physical effects.This enables the development and testing of a system with a basephysical structure to support cell viability and influence cellbehavior. The hydrogel matrix can then be rendered progressively morepermissive by the use of appropriate covalently-coupled cell adhesion orextracellular matrix molecules.

The 3-D matrices of this invention may be further modified, preferablychemically modified, to include hexosamine residues. Carbohydrates maybe involved in cell adhesion. Dodd and Jessel, J. Exp. Biol., 124, 00.225-38 (1986).

In another preferred embodiment, the compositions of this invention maybe used in neural cell transplantation. The ability of biosynthetichydrogels to organize, support and direct neurite extension from neuralcells may also be useful for applications such as 3-D neural cellculture and nerve regeneration. The bioartificial extracellular matricesof this invention may potentially carry one or more of the several celladhesion molecules that have been identified to play an important rolein cell migration and neurite extension in the developing nervoussystem, including N-CAM and Ng-CAM (Crossin et al., Proc. Natl. Acad.Sci., 82, pp. 6942-46 (1985); Daniloff et al., J. Neurosci., 6, pp.739-58 (1986)), tenascin (Wehrle et al., Development, 1990, pp. 401-15(1990) and L1 (Nieke and Schachner, Differentiation, 30, pp. 141-51(1985)). Among extracellular matrix glycoproteins, laminin has beenshown to be one of the most potent inducers of neurite outgrowth invitro. It is a component of the Schwann cell basal lamina and is thoughtto be involved in axonal regeneration in vivo (Baron-Van-Evercooren etal., J. Neurosci. Res., 8, pp. 179-83 (1983); Manthorpe et al., J. Cell.Biol., 97, pp. 1882-90 (1983); Rogers et al., Dev. Biol., 113, pp.429-35 (1983).

LN has also been found to enhance attachment of many neural cell types(McCarthy et al., J. Cell, Biol., 97, pp. 772-77 (1983); Liesi et al.,J. Neurosci. Res., 11, pp. 241-51 (1984); Hammarback et al., J.Neurosci. Res., 13, pp. 213-20 (1985); Kleinman et al., Annals NY Acad.Sci., 580, pp. 302-10 (1990), increase the survival of sympathetic andseptal neurons (Edgar et al., EMBO, 3, pp. 1463-68 (1984); Pixley etal., J. Neurosci. Res., 15, pp. 1-17 (1986), and stimulate neuriteoutgrowth in many peripheral and central neurons (Baron Van Evercoorenet al., J. Neurosci. Res., 8, pp. 179-93 (1983); Manthorpe et al., J.Cell, Biol., 97, pp. 1882-90 (1983); Rogers et al., Dev. Biol., 113, pp.429-35 (1983); Steele et al., J. Neurosci. Res., 17, pp. 119-27 (1987).

LN and other ECM constituents influence neuronal development in both theperipheral and the central nervous systems. Hence, presentingECM-oligopeptide derivatized agarose gels to the regeneratingenvironment in 3-D may enhance nerve regeneration when introduced inappropriate animal models.

The complex glycoprotein and proteoglycan components of theextracellular matrix are thought to provide permissive pathways forneural cell migration and neurite extension during development.Cell-cell and cell-extracellular matrix (ECM) interactions appear toregulate various aspects of neuronal cell differentiation includingneural cell migration and neurite extension. Sanes, Ann. Rev. Neurosci.,12, pp. 491-516 (1989).

Anatomical studies of neural development show that the migratorypathways of pioneer neurons seem to consist of a 3-D ECM that isorganized into a network of fibrils and granules. The chemotropicattraction of neuronal growth cones from their target areas, coupledwith a permissive three dimensional maze comprised of ECM molecules likelaminin (LN) and fibronection (FH) and some cell free spaces filled withhighly hydrates hyaluronic acid, is thought to play an important role inthe development of embryonic nervous system.

Laminin (LN), an ECM molecule derived from basal lamina, promotesneurite outgrowth in a wide variety of neural cells including dorsalroot ganglia (DRGs) and PC12 cells, a cell line derived from a ratpheochromocytoma. Kleinman et al., Annals NY Acad. Sci., 580, pp. 302-10(1990).

In development, the pathways followed by neural crest cells and thegrowth cones of pioneer neural cells contain several ECM constituents,including fibronectin, laminin, tenascin, thrombospondin and hyaluronicacid. Perris and Bronner-Fraser, Comments Dev. Neurobiol., 1, pp. 61-83(1989).

Thus, in one embodiment the agarose matrix is derivatized with neuritepromoting agents and growth factors to specifically enhance neuriteextension in agraose gels. It has been shown that the activity of theneurite promoting protein laminin is enhanced after it is complexed withheparin sulfate proteoglycan which helps organize specific molecularinteractions more favorable for neuritic outgrowth.

Integration of transplanted cells into host tissue results from growthof transplanted neurons, and from regeneration of axons from hostneurons damaged during the transplantation, with the establishment of afunctional interface, including graft-host interconnections and synapticrelationships. Woerly et al., Cell Transplantation, 2, pp. 229-39(1993). The 3-D agarose matrices of this invention may serve as asupport for directed growth of axons. We prefer neural stem cellsisolated according to Weiss et al., PCT/CA92/00283, most preferablyhuman stem cells.

The physio-chemical environment of various cells and tissues in vitromay be tailored to evoke particular and specific responses from them.Specific ECM peptides may be important in determining the degree offacilitation or permissivity to neurite outgrowth from neural cells. Inparticular, the nature of the peptide presented in 3-D can influenceneurite extension. Further, cell types may be differently affected by achosen peptide.

Neurite extension from PC12 cells in two-dimensions is enhanced by theIKVAV (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3) fragment of LN(Tashiro et al., J. Biol. Chem., 264, pp. 16174-182 (1989)). PC12 cellspossess as 110 kDa cell surface receptor (Kibbery et al., Proc. Natl.Acad. Sci., 90, pp. 10150-53 (1993)) which has been postulated to be thebinding site for the IKVAV (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3)sequence.

In a specific embodiment, agarose gels are derivatized with anIKVAV-containing sequence (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3)to promote neurite extension in PC12 cells.

The presence of nerve growth factor (NGF) may be required for neuriteextension. See, e.g., Sephel et al., Biochem. Biophys, Res. Comm., 2,pp. 821-29 (1989). These growth factors may be incorporated into thechannel membrane (U.S. Pat. No. 5,011,486), or may be continuouslyprovided within the channel by seeding the channel with cells thatsecrete the desired molecules, or a slowly released polymeric insert.See, e.g., U.S. Pat. Nos. 5,156,844 and 5,106,627. Such methods overcomeproblems associated with short half lives of various of the growthfactors, and problems with non-continuous or uncontrolled delivery ofthese factors.

In another specific embodiment, agarose derivatized with anRGD-containing sequence (ArgGlyAsp; AA₂ -AA₄ of SEQ ID NO:2), anCDPGYIGSR-containing sequence (CysAspProGlyTyrIleGlySerArg; SEQ IDNO:1), IKVAV-containing sequence (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ IDNO:3) or a cocktail (PEPMIX) containing a mixture of all threesequences, was used to promote neurite extension in E9 chick dorsal rootganglia.

In another method of this invention, lamination of alternatingnon-permissive, permissive and non-permissive gel layers permits thecreation of 3-D cell growth or migration "tracts" in vitro. The agaroseconcentration for the non-permissive layer can be an agaroseconcentration greater than 1.25%, preferably 2.0% or greater.

In other embodiment, lamination of gel concentrations 2%:1%:2%, with the1% gel layer carrying neural cells, e.g., dorsal root ganglia, canfacilitate the creation of 3-D neural `tracts`. Fabrication of thedirected 3-D neural tracts of this invention can be achieved byphysically casting neurite-extension permissive and non-permissive gelsin a controlled manner. Such casting methods are known in the art.

The factors controlling nerve regeneration across a gap followingtransection injury are not fully understood. Regeneration of severednerves does not normally include proliferation of nerve cells. However,the injured nerve cell will extend neurities that growth distally andattempt to reenter the intact neurilemmal sheath of the distal portionof the severed nerve. Conventional techniques for repair involvealigning the severed ends of the fascicles. This manipulation andsuturing stimulates growth and/or migration of fibroblasts and otherscar-forming, connective tissue cells. The scar tissue prevents theregenerating axons in the proximal stump from reaching the distal stumpto reestablish a nerve charge pathway.

Various nerve guidance channels have been developed in attempts toovercome these problems. See, e.g. U.S. Pat. Nos. 5,030,225 and5,092,871. One critical event in regeneration across a gap insmooth-walled silicone elastomer tubes is the formation of a fibrincable bridge which serves as a scaffold for migrating cells andelongating axons. Schwann cells, axons, endothelial cells andfibroblasts subsequently enter the gap region and orient intoregeneration units, blood vessels and epineurial and perineurial tissue,respectively. Aebisher et al., Brian Research, 531, pp. 211-18 (1990).

The agarose hydrogel compositions of this invention may be useful innerve guidance channels. Such nerve guidance channels are well known inthe art. Synthetic guidance channels have been used as inert conduitsproviding axonal guidance, maintaining growth factors, and preventingscar tissue invasion. Permselective channels with a molecular weightcut-off of 50,000 daltons allowed regeneration of nerves in a mousesciatic nerve model. The regenerated nerves were characterized by fineepineurium and high numbers of myelinated axons. Aebischer et al., "TheUse Of A Semi-Permeable Tube As A Guidance Channel For A TransectedRabbit Optic Nerve", In Gash & Sladek [Eds] Progress in Brain Research,78, pp. 599-603 (1988).

Permselective channels may support regeneration by allowing inwardpassage of nutrients and growth or trophic factors from the externalhost environment, while preventing the inward migration of scar-formingcells. Cells participating in the wound healing phenomena are known torelease various peptide growth factors. Several of these factors havemolecular weights in the range of 10-40,000 daltons. For example,activated macrophages secrete numerous growth factors, including NGF,bFGF, and apolipoprotein E.

Schwann cells distal to the nerve injury express low affinity NGFreceptors, as well as apolipoprotein B and E receptors. Binding ofapolipoprotein E to these receptors may enhance lipid uptake which caneventually be used in remyelination. Aebischer et al., Brain Research,454, pp. 179-87 (1988). Appropriate choice of the molecular weightcut-off for the permselective channels will allow retention of laminin(a high molecular weight glycoproptein) within the nerve guidancechannel. Similarly, blood vessels located in the proximal nerve stumpmay supply high molecular weight serum molecules such as fibronection orglycopropteins that have supported neuronal survival and neuriteextension in vitro. Aebischer et al., Brain Research, 454, pp. 179-87(1988).

The nerve guidance channels of the present invention include animplantable, biocompatible tubular permselective membrane havingopenings to receive the severed nerve. The lumen of the membranepreferably has a diameter ranging from about 0.5 mm to about 2.0 cm, topermit the nerve to regenerate through it. The thickness of the membranemay range from about 0.05 to about 1.0 mm. In some embodiments themembrane has a molecular weight cut-off of about 100,000 daltons orless. The membrane is preferably impermeable to fibroblasts and otherscar-forming connective tissue cells. Additionally, the membrane may becomposed of a biodegradable material. An agarose matrix is disposed inthe lumen of the nerve guidance channel. The agarose concentrationshould range between 0.5 to 1.25%, preferably 1.0%. The average gel poreradius can vary between 120 to 290 nm, and is most preferablyapproximately 150 nm.

The optimal concentration of agarose gel for use as a regenerationmatrix will vary according to the intended use of the matrix. Theoptimal concentration for in vitro use may not be optimal for the invivo milieu. Neurite outgrowth in agarose gels is strongly dependentupon the pore size of agarose gels. Syneresis at the channel mid-pointcould alter the pore size of agarose gels enough to inhibit regenerationand therefore result in the absence of nerve cable in the mid-portion ofthe regeneration nerve bundle. It is important to account and ifpossible, correct for syneresis of the gel at channel mid-point. Thismay be overcome by two strategies. One, the use of more dilute agarosegels to fill the channels may accommodate syneresis in the middle andstill retain the pore size of gel at the channel midpoint to rangespermissible for neurite extension. Second, the use of a rough innermembrane of the channel may serve to prevent the fibroblasts inducedsyneresis of the gel inside the guidance channel (Aebischer et al.,Brain Research, 531, pp. 21-18 (1990)).

In one method of repairing a severed nerve according to this invention,the cut ends of the nerve are placed in proximity with each other withinthe lumen of the tubular guidance channel. The cut ends of the nerve aregently drawn into the channel by manual manipulation or suction. Thenerve ends may be secured in position without undue trauma by sutures,or using a biocompatible adhesive, e.g., fibrin glue, or by frictionalengagement with the channel.

In addition to the agarose matrices of the present invention, the lumenof the channel may be "seeded" with a substance that protects, nurtures,and/or enhances nerve growth therethrough. Useful substances includebiologically active factors, such as nerve growth factors, brain derivedgrowth factor, basic fibroblast growth factor, acidic fibroblast growthfactor, or active fragments thereof. Alternatively, the lumen may beseeded with nerve-associated glial cells, such as Schwann cells. Thesegrowth factor cells and other nutrients may be provided within the nerveguidance channel as described supra.

The bioartificial ECMs of this invention may also carry one or more ofthe several cell adhesion molecules that have been identified to play animportant role in cell migration and neurite extension in the developingnervous system, including N-CAM, Ng-CAM (Crossin et al., Proc. Natl.Acad. Sci., 82, pp. 6942-46; Daniloff et al., J. Neurosci., 6, pp.739-58 (1986); tenascin (Wehrle et al., Development, 1990, pp. 401-15(1990) and L1 (Nieke et al., Differentiation, 30, pp. 141-51 (1985).

Other useful factors include cAMP, or analogs thereof, including 8-bromo8-bromo cAMP or chlorophenythio cAMP. See, e.g., U.S. Pat. Nos.5,030,225 and 5,011,486.

The nerve guidance channels of this invention may additionally be seededwith Schwann cells. Schwann cells resident in the peripheral nerve trunkplay a crucial role in the regenerative process. Schwann cells seeded inpermselective synthetic guidance channels support extensive peripheralnerve regeneration. Schwann cells secrete laminin, which possessesneurite-promoting activity in vitro. See, e.g., Aebischer et al., BrainResearch, 454, pp. 179-87 (1988). The Schwann cells are preferablylongitudinally oriented along the guidance channel. This can be achievedby thermal manipulation of the agarose gel to orient the poreslongitudinally, using methods well known in the art.

Insulin-like growth factor 1 (IGF-1) may also be useful in increasingthe rate of regeneration of transected peripheral nerves and to decreasethe persistence of permanent nerve function deficiency. See, e.g., U.S.Pat. No. 5,068,224.

Preferably the permselective membrane is fabricated to be impermeable tosome of these substances so that they are retained in the proximity ofthe regenerating nerve ends. See, e.g., Aebischer, U.S. Pat. No.5,011,486.

Briefly, various polymers and polymer blends can be used to manufacturethe nerve guidance channel. Polymeric membranes, forming the nerveguidance channel may include polyacrylates (including acryliccopolymers), polyvinylidenes, polyvinyl chloride copolymers,polyurethane, polystyrenes, polyamides, cellulose acetates, cellulosenitrates, polysulfones, polyphosphazenes, polyacrylonitriles,poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymersand mixtures thereof.

The membranes used in the nerve guidance channels of this invention maybe formed by any suitable method known in the art. One such methodinvolves coextrusion of a polymeric casting solution and a coagulant asdescribed in Dionne, WO 92/19195 and U.S. Pat. Nos. 5,158,881, 5,283,187and 5,284,761, incorporated herein by reference.

The jacket may have a single skin (Type 1, 2), or a double skin (Type4). A single-skinned hollow fiber may be produced by quenching only oneof the surfaces of the polymer solution as it is co-extruded. Adouble-skinned hollow fiber may be produced by quenching both surfaces othe polymer solution as it is co-extruded. Typically, a greaterpercentage of the outer surface of Type 1 hollow fibers is occupied bymacropores compared to Type 4 hollow fibers. Type 2 hollow fibers areintermediate.

The jacket of the nerve guidance channel will have a pore size thatdetermines the nominal molecular weight cut off (nMWCO) of thepermselective membrane. Molecules larger than the nMWCO are physicallyimpeded from traversing the membrane. Nominal molecular weight cut offis defined as 90% rejection under convective conditions. Typically theMWCO ranges between 50 and 200 kD, preferably between 50 and 100 kD.

A preferred nerve guidance channel according to this invention forpromoting regeneration of peripheral nerves across large gaps, includesan agarose matrix optimized in the regeneration environment to suit there-growth of a particular nerve, the presence of Schwann cells seeded inthe lumen of the channel, and the local release of growth factors fromthe wall of the guidance channel.

In one embodiment, agarose hydrogels are used as a carried to presentthe laminin derived oligopeptide CDPGYIGSR (CysAspProGlyTyrIleGlySerArg;SEQ ID NO:1) to the site of nerve injury in an attempt to enhance nerveregeneration. Dorsal root ganglia have been shown to be responsive toCDPGYIGSR (CysAspProGly TyrIleGlySerArg; SEQ ID NO:1) in vitro and showthe greatest enhanced neuritic spread and neurite outgrowth compared toother fragments derived from laminin.

Compared to the ventral roots, transected dorsal roots have a limitedregeneration through polymeric guidance channels across a nerve gap of 4mm in adult rats after 4 weeks. McCormack et al., J. Comp. Neurol., 313,pp. 449-56 (1991), Rat hind limb sural nerves have been shown to containmainly sensory nerves with only 5% to 10% motor fibers Peyronnard etal., Muscle and Nerve, 5, pp. 654-60 (1982). Dorsal root and sural nervetransection enables comparison of regeneration between a sensory nervecable close to the central nervous system, i.e., dorsal root, and onethat is more peripheral, i.e., the sural nerve.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of thisinvention in any way.

EXAMPLES Example 1 Characterization of Agarose Hydrogel Matrices

E14 striatal cell assay

Striata were removed from 14-day-old rat embryos and mechanicallydissociated with a fire-narrowed Pasteur pipette in serum-free medium.Different concentrations of agarose gels in the range of 0.5% to 5.0%were made in phosphate buffered saline pH 7.4. Gel solutions weresterilized by passing them through sterile 0.2 micron filters. IsolatedE14 striatal cells were mixed into the gel solutions at room temperaturein 1 ml syringes, each containing 300 μl of appropriate agarose gelconcentration. The cell-gel solution mixture was then decanted into 48well Costar tissue culture dishes. The dishes were cooled to 4° C. forthe cell-gel solution mixture to gel, suspending the striatal cells in3-D. One ml of a 1:1 mixture of DMEM and F12 nutrient (Gibco)supplemented with 5% fetal calf serum, glucose (33, M), glutamine (2mM), sodium bicarbonate (3 mM), HEPES buffer (5 mM, pH 7.4), insulin (25μg/ml), transferrin (100 μg/ml), putrescine (60 μg/ml.), progesterone(20 nM) and sodium selenite 30 nM) (all from Sigma) was added to the topof the gels. The gels were cultured in an incubator at 37° C. in 95%air, 5% CO₂ and 100% humidity. Striatal cells were also suspended in100% Matrigel® at 4° C. and quickly decanted into 48 well Costar dishesand cultured in the manner describes above.

The percent striatal cells expending neurites of every 500 cellssuspended in 3-D was measured for the agarose gel range of 0.5% to 5.0%(wt/vol) at 24, 48 and 72 hours in culture. Meurite extension wasobserved under light microscopy using a Zeiss Axiovert MC100 phaseinversion microscope. All neurites whose length was greater than twicethe striatal cell body diameter were counted.

E14 striatal cells extended neurites in 3-D in 1% agarose gels. Neuriteextension from E14 striatal cells in 1% agarose gels was comparable toneurite extension in 100% Matrigel® after 72 hours in culture. E14striatal cells extended neurites in the 0.5% to 1.25% gel range but didnot extend neurites above a threshold concentration of 1.25% (wt/vol).

Chick dorsal root ganglion assay

Dorsal root ganglia were dissected from E9 chick embryos by a standardprotocol. Chick embryos were immobilized in a prone position and a 3 mmlong incision made on either side of the spine exposed DRGs forexplanation. The DRGs were added to a 300 μl solution of 1% agarose in a1 ml syringe at room temperature, mixed gently and decanted intocustom-built 9×9 mm cube shaped glass culture dishes. The 9×9 mm disheswere then placed at 4° C. for agarose to gel, trapping DRGs in 3-D. Thecubic glass dish enables visualization of the X-Z axis, which is theplane perpendicular to the bottom of the culture dish. Three hundredmicroliters of 1% Ag-Plain gel solution was drawn into a 1 ml syringe,and the DRGs added to the gel solution with a micropipette. Theganglion-gel solution mixture was then decanted into the 9×9 dishes andcooled at 4° C. for 5 min, trapping the DRGs in the gel. Each well hadtwo DRGs suspended int it. One ml of DMEM/F12 medium (Gibco) containing10 ng/ml 2.5 s nerve growth factor (Sigma), glucose (0.3%),penicillin-streptomycin (1%), L-glutamine (200 mM), KCI (1.5 mM),insulin (0.08 mg/ml), transferring (10 mg/ml), putresine (6 mM),and 5%fetal calf serum was added to the top of the gels. The cultures weremaintained in an incubator at 37° C. with 100% humidification, 95% airand 5% CO₂. The cubic glass dish was flipped on its side after 6 days inculture, exposing the X-Z axis for analysis with light microscopy.

For neurite extension analysis along the X-Y axis, which is the planeparallel to the bottom of the culture dish, DRGs were suspended in plainagarose, agarose-GRGDSP (GlyArgGlyAspSerPro; SEQ ID NO:2),agarose-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1),agarose-x-IKVAV(IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3)-x,agarose-PEPMIX and agarose-GGGGG (GlyGlyGlyGlyGly; SEQ ID NO:4) gels instandard 48 well Costar tissue culture dishes in the manner describedabove for the cubic glass culture dishes. At least 6 ganglia wereanalyzed for each type of agarose gel. The final concentration of thecell-gel solution mixture was 0.83% (wt/vol) and the total cell-gelvolume in each culture well was 300 microliters.

Chick DRGs extended neurites in 1% agarose gels in both the X-Y and X-Zplanes when cultured in the cusion designed 9×9 mm glass cubic dishesafter 6 days in the presence of NGF. DRGs extended long and tortuousneurites along the X-Y axis and the X-Z axis demonstrating the 3-Dnature of neurite extension in agarose gels.

Gel Characterization

a. Hydraulic permeability: Gel blocks of different concentrations, eachof thickness 0.5 cm and radius 1 cm, were mounted on a custom-builtwater column. Each block was subjected to a known hydraulic pressure,typically a 100 cm high H₂ O column yielding approximately 24525dynes/cm². The hydraulic permeability per unit time for a givenhydraulic pressure was measured for the various gel concentrations. Theaverage pore radius of the gel concentration range 0.5% to 5.0% wascalculated as described by Refojo et al., J.Appl.Poly.Sci., 9, pp.3417-26 (1965) using the hydraulic permeability.

The average pore radius, calculated from the hydraulic permeabilitymeasurements of the various agarose gels, decreased exponentially as thegel concentration increased (FIG. 1). E14 striatal cells did not extendneurities beyond a threshold agarose gel pore radius of 150 nm. Theslope of the curve depicting pore radius was steep between gelconcentrations of 1% and 2% indicating a strong dependence of pore sizeon gel concentration.

b. Scanning Electron microscopy (SEM): Agarose gels in the range 0.5% to2.0% were freeze-dried, mounted on aluminum stubs, coated with gold andanalyzed under a Joel 35M scanning electron microscope. Representativesections of the scanning electron micrographs were selected forevaluating the morphology and size of the pores.

Scanning electron micrographs of different concentrations of agarosegels revealed an open-cell morphology.

c. Electron microscopy (ESEM): Agarose gels of the concentration range0.5% to 2.5% were analyzed with an environmental scanning electronmicrograph (Electroscan ESEM, type E3) under partially hydrated statesto qualitatively asses gel pore morphology.

A decline in gel cavity radius was noted with increasing gelconcentration. However, the nature and quality of images obtained withthe ESEM allowed only qualitative conclusions on gel pore size to bedrawn with confidence.

d. Gel electrophoresis: The electrophoretic mobility of insulin (Mw5,700), bovine serum albumin (Mw. 66,000; radius 140 Angstroms) andbovine thyroglobulin (Mw. 669,000) in 1%, 2% and 4% agarose gels wasmeasured under a constant electrophoretic voltage gradient. Twenty ml ofthe appropriate agarose gel concentration was poured into a DANAPHORmodel 100 mini gel electrophoresis apparatus (Tectate S. S.,Switzerland) with platinum electrodes. The proteins insulin, albumin andthyroglobulin were then subjected to a constant electrophoretic voltagegradient of 1 to 12V. The protein electrophoretic mobility was measuredin the 1%, 2% and 4% agarose gels by measuring distance traveled perunit time. The relative electrophoretic velocity was then calculatedafter taking into account the isoelectric points of the differentproteins, the voltage employed and the time of exposure to enableelectrophoretic mobility comparisons of insulin, albumin thyroglobulinin the agarose gels.

The relative electrophoretic mobility of the globular proteins insulin,albumin and thyroglobulin, fell with increasing gel concentration. Theelectrophoretic mobility of insulin and albumin decreased by relativelysmall percentage in 2% agarose gels compared to 1% gels i.e., by 5.7%and 2.9% respectively. In contrast, the relative electrophoreticmobility was attenuated by 33.3% in 2% agarose gels relative to that in1% agarose gels for the large molecular weight globular protein, bovinethyroglobulin.

Laminated Gels

A 3 mm thick, cell-free layer of non-permissive 2% agarose gel solution,a 1 mm layer of permissive 1% gel solution with chick DRGs mixed in it,and an additional 3 mm, cell-free layer of non-permissive 2% agarosesolution were serially cooled and gelled in a custom designed 9×9 mmcubic glass dishes. The dishes were then turned on their side exposingthe X-Z axis and analyzed under light microscopy after 6 days inculture. The gel interfaces were examined for neurite cross-over fromone gel layer to the other.

Chick DRGs suspended in the permissive 1% agarose gel layer extendedneurites only in the 1% gel and did not cross-over the 1%:2% gelinterface. In comparison, many neurites were able to cross-over acontrol gel interface of 1%:1% agarose gel. By experimental design, allthe neurites encountering the interfaces were extending in the X-Z axisof the gel, perpendicular to the bottom of the culture dish.

Example 2

A Bioartificial ECM of Oligopeptide-Derivatized Agarose

Preparation of Agarose Gels

One percent (wt/vol) hydroxyethylated agarose (SeaPrep®, FMC Corp.Rockland, Me.) gels were prepared by dissolving agarose in phosphatebuffered saline (PBS) at pH 7.4. The gel solutions were passed through a0.2 micron filter for sterilization. Gel solutions were then placed at4° C., allowed to gel, and stored at 4° C. until they were derivatizedwith peptides.

Immobilization of Laminin Oligopeptides

Agarose gels were derivatized with 1,1 carbonyldiimidazole (CDI) (Sigma)using a modified version of the protocol described by Hearn, MethodsEnzymol., 135, pp. 102-17 (1987). See FIG. 2 for schematic. Three tofour ml gel blocks of 1% agarose were dehydrated by repeated washes inacetone followed by acetone which was dried under 4 Angstrom molecularsieves (Sigma). A 150 mg/25 ml CDI solution prepared in dry acetone wasadded to the acetone washed agarose gels (5 ml/3 g gel block). Theactivation reaction was allowed to proceed for 9 min with gentleagitation. Gels were then washed five times with dry acetone for 6 minper wash to remove unbound CDI.

CDI activated gels were then exposed to various oligopeptides dissolvedin 100 mM sodium bicarbonate buffer solution at pH 8.5 at aconcentration of 0.6 mg/ml. Peptide coupling reaction was allowed toproceed for 36 hr under gentle agitation.

The gels were then washed thoroughly with PBS for 48 hr, furtherquenched in sodium bicarbonate for 2 hr at room temperature, lyophilizedand re-dissolved to the desired gel concentration of 1.0%.

The peptides used were GRGDSP (GlyArgGlyAspSerPro; SEQ ID NO:2) (Teliospharmaceuticals, San Diego, Calif.), CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1), the 19-mer sequenceCSRARKQAASIKVAVSADR (CysSerArgAlaArgLysGlnAlaAlaSerIleLysValAlaValSerAlaAspArg; SEQ ID NO:3), andx-IKVAV-x containing sequence (s-IleLysValAlaVal-x; AA₁₁ -AA₁₅ of SEQ IDNO:3) (Anawa, Wagen, Switzerland) and as a control, GGGGG(GlyGlyGlyGlyGly; SEQ ID NO:4) (Sigma). A cocktail of the threeaforementioned peptides (PEPMIX) was also immobilized to the hydrogelbackbone at a concentration of 2 mg each in a total of 5 ml buffersolution.

The 1'1 carbonyldiimidazole coupling reaction used for immobilizingpeptides to the agarose gels was verified by binding radiolabelled ¹⁴ Cglycine as a model amino compound for the various peptides. Gels whichwere not activated with CDI, but exposed to 14C glycine were used ascontrols. Beta counts for CDI activated and CDI deficient gels werecounted with a β counter (LKB Wallac, 1217 RackBeta liquid scintillationcounter) after dialyzing for 12 days.

CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) was labelled with¹²⁵ I using lactoperoxydase (Sigma). Ten μl of CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) solution in 0.4M sodiumacetate solution (5 μg/10 μl of sodium acetate), 1 millicurie of ¹²⁵ I(Amersham Radio Chemicals) and 10 μl of hydrogen peroxide (H₂ O₂ ; 1 in20,000 parts). The reaction was allowed to proceed for 1 min and stoppedwith 500 μl of 0.1M sodium acetate solution.

Radiolabelled CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) waseluted with 20% to 50% methanol in an octodesasilicic acid (ODS) gelcolumn (Shandon Scientific Ltd., Chesire England). ¹²⁵ I CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) was coupled to agarose gelsin the manner described above. Gamma counts were analyzed with a Packardautoscintillation spectrometer (5416) after 5 days of washing to removeunbound peptide.

Beta counts of ¹⁴ C glycine bound to CDI derivatized agarose revealedthat up to 37 μg of glycine was retained per gm of gel after 12 days ofdialysis. CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) wassuccessfully radiolabelled with ¹²⁵ I and the peak elution is ODS gelswas the 40% methanol. In the presence of CDI, 2.39 times more ¹²⁵ Ilabeled CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) wasretained in agarose gels compared to gels without CDI after 5 days ofdialysis of the gel. The short half-life period of ¹²⁵ I preventedfurther washing even though the gamma counts in the CDI deficient gelwere failing more rapidly with each successive wash compared to thecounts in CDI activated agarose gel.

Gel Characterization

Gel porosity of underivatized agarose gels and glycine coupled agarosewas determined as described in Example 1. The average pore radius of thegels were determined to be 310 nm for a 0.5% underivatized agarose geland 360 nm for a 0.5% glycine coupled agarose gel using the water columnfor hydraulic permeability measurements.

Dorsal Root Ganglion Assay

DRGs were dissected from E9 chick embryos by a standard protocol, asdescribed in Example 1.

Neurite extension was analyzed qualitatively along the X-Y axis using aZiess axiovert MC100 TV light microscope at days 2, 4 and 6. Cellviability of both cell types was assessed by a fluorescein diacetate(FDA) assay at day 6. For the DRG study, the ganglionic cell body area(GCBA), and the total ganglionic spread area (TGSA) defined as themaximum area covered by the ganglion and its neurites, were measured inthe X-Y plane using an NIH Image 1.47 software package. The ratio ofTGSA/GCBA, defined as the total neuritic spread of the ganglia, wascalculated. The length of five of the longest neurites extended by theDRGs was also measured to assess neurite extension.

Agarose hydrogels supported neurite outgrowth from DRGs in both X-Y andX-Z planes, demonstrating the 3-D character of neurite outgrowth inagarose gels. Fluorescein diacetate assay showed viable DRG neuronsafter 6 days in culture in all agarose gels used.

DRG neurons extended neurites in all of the agarose gels when examinedin the X-Y plane in 48 well Costar dishes including Ag-Plain andAg-CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1).

The total ganglionic spread area/ganglionic cell body area ratio(TGSA/GCBA) was calculated at days 2, 4 and 65 to account for thevariance in the size of the dorsal root ganglia after dissection and asa measure of the total neuritic spread in the gels (FIG. 3). Compared toAgPlain gels, Ag-PEPMIX gels showed significantly greater neuriticspread at all measured time points (p<0.05) while CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) had significantly greaterneuritic spread only at day 6 (p<0.05). In contrast, IKVAV-derivatized(IleLysValAlaVal; AA₁₁ -A₁₅ of SEQ ID NO:3) gels had significantlylesser neuritic spread at all measure time points while GRGDSP(GlyArgGlyAspSerPro; SEQ ID NO:2) had significantly lower neuriticspread only at days 2 and 4 (p<0.05), compared to AgPlain gels at allmeasured time points.

CDPGYIGSR (GysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) and PEPMIXderivatized agarose gels supported significantly longer neurites thanunderivatized agarose gels (p<0.05) at day 6 (FIG. 4). In contrast,IKVAV-derivatized (IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3) agarosegels inhibited neurite outgrowth at days 2, 4 and 6 (p<0.05). Ag-GGGGG(GlyGlyGlyGlyGly; SEQ ID NO:4) gels had a significantly lower (p<0.05)neurite length compared to Ag-Plain at day 2 but there was nostatistical difference in neurite length between the two gels at days 4and 6. In all the agarose gels, neurites extended three dimensionallyincluding the X-Z plane. The neurites were long and tortuous, extendingup to 1600 microns at day 6 in CDPGYIGSR-derivatized(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) gels.

PC12 Cell Assay

PC12 cells (American Type Culture Collection, Rockville, Md.) were grownin Gibco's RPMI 1640 with 10% fetal calf serum, 5% horse serum,L-glutamine (200 mM) and 1% penicillin-streptomycin. They were primed atthe tenth passage with 10 ng/ml 1.5 s nerve growth factor (NGF) for 48hr prior to use. Primed PC12 cells were then mixed in the variousagarose gel solutions at a density of 50,000 cells per ml using a methodsimilar to the one described above for DRGs. The cell-gel solutionmixtures were poured into 48 well Costar tissue culture dishes andallowed to gel by cooling 4° C. One ml of PC12 medium was added to thetope of the gels along with 10 ng/ml of NGF. The cultures were placed inan incubator at 37° C. with 100% humidification, 93% air and 7% CO₂.

The percentage of PC12 cells extending neurites in the various agarosegels was assessed by selecting optical cylindrical sections of the gelsfor analysis at a magnification of 200× under light microscopy. Opticalcylindrical sections were chosen by serially moving the microscopevisual field from the center of the dish to the side along parallelpaths. At least nine hundred PC12 cells were examined for neuriteextension in each well and at least 6 wells of each type of agarose gelwere analyzed. The inclusion criterion for a positive count was aneurite longer than on PC12 cell body diameter. Two sided Student t-testwas employed to determine statistical significance with p<0.05considered to be significant.

PC 12 cells were viable as evidenced by an FDA assay in all gels exceptAg-GGGGG (GlyGlyGlyGlyGly; SEQ ID NO:4). PC12 cells also extendedneurites in all gels after 6 days in culture except in Ag-GGGGG(GlyGlyGlyGlyGly; SEQ ID NO:4) gels. However, the percent PC12 cellsthat extended neurites depended upon the type of gel used. At allmeasured time points, the laminin oligopeptide derivatized agarose gelssupported significantly greater percent neurite extension (p<0.05) thanunderivatized Ag-Plain gel. Neuritic extension inAg-x-IKVAV(IleLysValAlsVal; AA₁₁ -AA₁₅ of SEQ ID NO:3)-x gels wassignificantly higher (p<0.05) compared to the Ag-GRGDSP(GlyArgGlyAspSerPro; SEQ ID NO:2), Ag-CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1), Ag-PEPMIX and underivatizedagarose gels at days 4 and 6 (FIG. 5).

In the absence of NGF, no neurite extension was observed in any of theagarose gels. No measurable difference in the neurite length wasobserved between the Ag-plain and the various derivatized agarose gels(data not shown). The overall percent of PC12 cells extending neuritesin the various derivatized agarose gels, IKVAV-derivatized(IleLysValAlaVal; AA₁₁ -AA₁₅ of SEQ ID NO:3) gels including, was lowerthan the percent cells extending neurites in 100% Matrigel® or 1.2 mg/mlVitrogen® gels (36.5% and 32.8% respectively at 4 days).

Example 3

The effect of derivatized agarose gels on the regeneration of transectedrat spinal dorsal roots was evaluated by using 6 mm long polymerguidance channels filled with CDPGYIGSR (CysAspProGlyTyrIleGlySerArg;SEQ ID NO:1)--agarose to bridge a 4 mm gap in a transected dorsal rootmodel. After 4 weeks, significantly greater numbers of myelinated axonswere observed in the channels filled with CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1)--agarose gels compared to channels filledwith underivatized agarose gels.

Guidance Channels

Guidance channels were fabricated from acrylonitrile-vinylchloride(PAN/PVC) copolymers by wet-jet wet spinning. Cabasso, I., InEncyclopedia of Chemical Technology, 12, pp. 492-517 (1978); Aebischeret al., Biomaterials, 12, pp. 50-56 (1991). The channel consisted of asmooth inner and outer skin with a molecular weight cut-off 50,000 Da,with an open trabecular network in between, which provided thestructural support for the channel. Channels of internal diameter 0.8 mmand 0.5 mm were fabricated for the spinal root and sural nerve modelrespectively.

CDPGYIGSR Derivatized Agarose Gels

One percent (wt/vol) agarose gels were derivatized with LN oligopeoptideCDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) as described inExample 2. PAN/PVC guidance channels were sterilized by storing in 70%ethanol overnight. Guidance channels were filled with CDPGYIGSR(CysAspProGlyTyrIle GlySerArg; SEQ ID NO:1) derivatized andunderivatized agarose gel solutions and the ends of the channel weresealed with heat to prevent leakage of gel solution. The channels werethen cooled to 4° C. to allow agarose solutions to gel. After agarosesolutions "gelled" inside the channels they were cut to a standardlength of 6 mm and 10 mm for the dorsal root and sural nerve implantsrespectively.

Animal Model and Guidance Channel Implantation

Adult male albino rats (Wistar) weighing 180 to 220 gm were anesthetizedby an intrapertioneal injection of pentobarbital (60 mg/kg). The dorsaloperative site was shaved and swabbed with an iodophore (Butadiene). A 5mm dorsal midline incision was made using the iliac crests as landmarks.Retraction of paraspinous muscles exposed L2-L6 vertebrae. Bilaterallaminectomies were performed on L2 to L5 to expose the spinal cord. Thespinal roots were exposed by a midline incision on the dura mater. Usinga blunt nerve hook the dorsal root exiting the L4 vertebra wasidentified and a 2 mm section removed. Six millimeter long guidancechannels carrying underivatized agarose and CDPGYIGSR-derivatized(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) agarose were used to bridgethe 2 ends of the nerve using a 10-0 monofilament nylon suture (Ethicon)(See FIG. 6 for schematic). Five rats received channels filled withunderivatized agarose and six animals received channels filled withCDPGYIGSR-derivatized (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1)agarose.

For the sural nerve model, the hind limb operative site was shaved andcleaned with iodophore. "L" shaped incisions were made on the left hindlimbs of 180-220 g albino rats (Wistar) along and dorsal to the femurand continuing past the knee. The gluteus maximus muscle was retractedand the sciatic nerve was exposed. The sural branch of the sciatic nervewas identified, followed down beneath the knee joint, and a 2 mm longpiece of the nerve was transected. The ends of the nerve were thenbridged with guidance channels which were filled with either saline orunderivatized agarose or CDPGYIGSR-derivatized(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) agarose. Five animalsreceived saline filled channels, five received agarose plain channelsand four received CDPGYIGSR (CysAspProGly TryIleGlySerArg; SEQ IDNO:1)-agarose filled channels. All animals were housed in an controlledenvironment with 12 hour on-off light cycles. They received food andwater ad libitum.

Implant Retrieval and Evaluation

Four weeks post-implantation, the animals were deeply anesthetized withan intra-pertioneal injection of sodium pentobarital and transcardiallyperfused with 200 ml of heparinized physiologic saline followed by 250ml of a cold 4% paraformaldehyde 2.5% gluteraldehyde solution inphosphate buffered saline at pH 7.4. The operative site was reopened,and the guidance channel retrieved. The specimens were post-fixed,dehydrated and embedded in glycomethacrylate. The cable cross-sectionalarea, the number of myelinated axons at 1.5 mm, 2 mm and 3.5 mm of thedorsal root channel were analyzed with NIH software 1.47 interfaced witha Zeiss Axiovert Mc100 microscope. The proximal nerve stump was definedto be at the 0 mm point and the distal nerve stump at the 4 mm point ofthe 4 mm nerve gap in dorsal roots. For the sural nerve implants, thecable cross-sectional area and the number of myelinated axons at 2 mmpoint along the channel was evaluated on 6 micron semi-tin sections. Allsections were stained with osmium tetraoxide and cresyl violet.

Dorsal Root Regeneration

Semi-thin cross-sections along the length of the guidance channel showedthat myelinated axons were present all along the 4 mm nerve gap.Histological sections of guidance channels filled with agarose gelscarrying regenerated dorsal roots showed doughnut shaped, centrallylocated nerve cables. Tissue reaction to the PAN/PVC polymer consistedof multi-nucleate giant cell and connective tissue infiltration.Neovascularisation was evident in the midst of regenerated nervoustissue along the Schwann cell infiltration. Light micrographs ofregenerating dorsal root cables at 4 weeks post-implantation at themid-point of guidance channels showed comparatively more nervous tissuein channels containing CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ IDNO:1) gels relative to channels with underivatized agarose. At themide-point of the 4 mm nerve gap, the channel filled with AgCDPGYIGSR(CysAspProGly TyrIleGlySerArg; SEQ ID NO:1) had a significantly greater(p-0.05) number of myelinated axons compared to the channels withagarose plain gels (see FIG. 7). The number of myelinated axons at themidpoint in the agarose plain filled channels were comparable to thosein saline filled channels described by McCormack et al., J.Comp.Neurol., 313, pp. 449-56 (1991). CDPGYIGSR-derivatized (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) agarose gels had a significantly higherdensity (p<0.05) of myelinated axons at 0.5 mm and 2.0 mm along thechannel at 4 weeks postimplantation (FIG. 8). The density of myelinatedaxons is defined as the number of myelinated axons per 10⁵ squaremicrons of cable area.

Sural Nerve Regeneration

All of the 10 mm long guidance channels implanted to bridge transectedsural nerves were kinked due to flexion at the rats' knee-joint.Histological evaluation of nerves proximal to the kink showedregenerated cables located at the center of the channel with myelinatedaxons. All cables present in the channel distal to the kink contained afibrotic cable but no myelinated axons. Almost all of the kinks occurredbetween 2-4 mm into the nerve gap. Therefore only the 2 mm point wasanalyzed for myelinated axons in this study. Light micrographs ofregenerating sural nerves, 4 weeks post-implantation at the 2.0 mm pointin polymer guidance channels filled Ag-CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) showed relatively highernumbers of nerve fascicles and neovascularization than in the otherguidance channels. At 2 mm into the 8 mm nerve gap, guidance channelsfilled with AgCDPGYIGSR (CysAspPro GlyTyrIleGlySerArg; SEQ ID NO:1) gelshad a significantly greater number (p<0.05) of myelinated axons comparedto either the saline filled channels or agarose plain filled channels.See FIG. 9. At this point, the density of myelinated axons in theCDPGYIGSR-derivatized (CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) agarosechannels was also significantly greater (p<0.05 ) than the density ofaxons in the agarose-plain and saline-filled channels (FIG. 10). Whenthe number of myelinated axons was compared to the number present in anormal control sural nerve, at 4 weeks, the average number of myelinatedaxons regenerated across the saline filled channel was 12% of controlsural nerve, 13% of control nerve for AgPlain filled channels and 40% ofcontrol sural nerve for AgCDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ IDNO:1) filled channels.

Transected rat dorsal roots regenerate across a 4 mm gap after 4 weeksin semipermeable guidance channels filled with agarose gels derivatizedwith LN oligopeptides. This is in contrast to the limited regenerationobtained by earlier experiments in the same model (McCormack et al., J.Comp. Neurol., 313, pp. 449-56 (1991) using channels filled with salineonly. Though there was no significant difference in the number ofmyelinated axons 0.5 cm into the nerve gap, channels filled withCDPGYIGSR (CysAspProGly TyrIleGlySerArg; SEQ ID NO:1) derivatizedagarose gels had significantly greater myelinated axons at the mid pointof the channel compared to channels filled with agarose plain. Thisobservation suggests a faster rate of nerve regeneration in gelsderivatized with the CDPGYIGSR (CysAspProGlyTyrIleGlySerArg; SEQ IDNO:1) compared to AgPlain gels.

The number of myelinated axons at the midpoint of the channel inunderivatized agarose gels was comparable to the number obtained in thesaline filled channels of McCormack. This demonstrates that agarose gelsare not inhibitory to regeneration in semipermeable channels as someother matrices like Matrigel® have been shown to be. See, e.g.,Valentini et al., Exp. Neurol., 98, pp. 350-56 (1987). CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) derivatized agarose gels alsohave a higher density of myelinated axons in the regenerated cablecompared to underivatized agarose gels at 0.5 and 2.0 mm nerve gappoints. This observation coupled with the data on numbers of myelinatedaxons along the channel, indicates that there is lesser fibrotic tissueper cable area in the derivatized agarose gels compared to underivatizedAgPlain gels.

The doughnut shape of the regenerated cable at the midpoint of thechannel points to possible syneresis of the agarose gels due to cellularactivity in the regeneration environment.

All of the 10 mm long guidance channels across the 8 mm sural nerve gapwere kinked distal to the point 2 mm into the nerve gap because of thechannel was located across the knee-joint of the rat. However, at the 2mm point, both the number and density of myelinated axons were greaterin channels filled with CDPGYIGSR-derivatized(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1) agarose compared to channelsfilled with underivatized agarose or saline.

Therefore in both the dorsal root model and in the more peripheral suralnerve model, CDPGYIGSR-derivatized (CysAspProGlyTyrIleGlySerArg; SEQ IDNO:1) agarose channels had greater numbers of myelinated axons in higherdensities per cable area compared to channels filled with underivatizedagarose or saline filled channels.

This data indicates the feasibility of developing a matrix designed toenhance nerve regeneration by coupling neurite promoting biomolecules toagarose hydrogels.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Cys Asp Pro Gly Tyr Ile Gly Ser Arg                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gly Arg Gly Asp Ser Pro                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Il - #e Lys Val Ala Val Ser      1               5   - #                10  - #                15               - - Ala Asp Arg                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Gly Gly Gly Gly Gly                                                      1               5                                                            __________________________________________________________________________

We claim:
 1. A bioartificial extracellular matrix comprising athree-dimensional high water content derivatized hydrogel matrix havinga hydrogel matrix core(a) wherein the hydrogel matrix is derivatizedthrough the matrix by covalent-immobilization of at least one celladhesive peptide fragment, homogeneously dispersed throughout thehydrogel matrix, and (b) wherein the hydrogel matrix has an average poreradius greater than 120 nm.
 2. The matrix according to claim 1, whereinthe amino acid sequence is SEQ ID NO:1.
 3. The matrix according to claim1, wherein the amino acid sequence is SEQ ID NO:2.
 4. The matrixaccording to claim 1, wherein the amino acid sequence is SEQ ID NO:3. 5.The matrix according to any one of claims 1 and 2-4, wherein thehydrogel matrix is a polysaccharide hydrogel matrix.
 6. The matrixaccording to claim 1, wherein the hydrogel matrix is an agarose hydrogelmatrix.
 7. The matrix according to claim 6, wherein the agaroseconcentration in the hydrogel matrix ranges between 0.5-1.25% (w/v) andthe hydrogel matrix has an average pore radius ranging between 120-290nm.
 8. The matrix according to claim 6, wherein the agaroseconcentration in the hydrogel matrix is 1.0% (w/v) and the hydrogelmatrix has an average pore radius of approximately 150 nm.